The long term goal of this project is to understand the molecular mechanisms by which ligand processing and receptor recycling are regulated during endocytosis. Although the hepatic asialoglycoprotein receptor (ASGPR) was the first endocytic, recycling receptor identified and has been well studied, important information about how this and other endocytic receptors function is still not known. In particular, the mechanism by which internalized ligand and receptor are segregated from each other is unknown. This is likely to be important in diseases with ASGPR defects, such as diabetes and alcoholism, because segregation can determine the efficiency of endocytosis. If this key step is inefficient, ligands will rebind to receptors and be nonproductively recycled to the cell surface, rather than being routed to lysosomes. In 1986 we hypothesized that endocytosis mediated by ASGPRs, and also other endocytic receptors occurs via two different cellular coated-pit pathways. This Two Pathway hypothesis has now been validated by others who isolated cell lines defective in only one of these two predicted pathways, the State 2 pathway. A second related hypothesis is that internalized State 2 ASGPRs undergo a transient inactivation and reactivation (I/R) cycle as they traverse their intracellular recycling route. I/R cycles would ensure the efficient operation of many endocytic receptors, since inactivated receptors could not rebind ligand during segregation. Both hypotheses apply to endocytic receptors in general. This laboratory has now reconstituted the ASGPR I/R cycle in permeable cells and discovered its molecular basis; covalent modification by fatty acids. The ligand-binding activity of State 2 ASGPRs is reversibly regulated by fatty acid acylation/deacylation of Cys residues. The role of fatty acylation of the two human subunits (HI & H2) in ASGPR structure and function will now be elucidated, and our hypotheses further refined. The specific aims are: l) To determine the fatty acylation status of Cys residues in mutant HI and H2 subunits of the human ASGPR. 2) To characterize the ability of stably transfected cell lines expressing Cys-mutant HI and H2 subunits to mediate ASGPR endocytic functions. 3) To characterize the modifying groups in the cytoplasmic and transmembrane domains of wildtype H1 and H2. 4) To analyze sequence determinants necessary for fatty acylation or deacylation of Cys36 and Cys57 in H1, the major subunit of the human ASGPR, and 5) To identify specific determinants in HI or H2 that direct ASGPRs into the State l or State 2 endocytosis pathways. Results from these studies will define, at the molecular level, the mechanisms that regulate normal ASGPR recycling and should lead to strategies for identifying and treating abnormal endocytic receptor regulation in human diseases.